The applicant is a specialist in infectious diseases with a long-term interest in tuberculosis and international health. His background in cellular immunology is the basis for the proposed research. He studied cellular immunology of T cells and dendritic cells at the NIH as Howard Hughes Research Scholar as well as protein biochemistry as an undergraduate at the University of Chicago. He has an excellent publication record from his previous research experiences at the NIH, in the laboratory of Robert Seder and from work performed in the laboratory of Thomas Hawn. His immediate goals are to study the mechanisms of TOLLIP regulation of the innate immune response. He will accomplish this using a number of specialized resources as part of a training program. First, his mentor (Dr. Thomas Hawn) has experience studying common genetic variation in large human cohorts that will be applied for the first time to TOLLIP. Second, his co-mentor (Dr. Alan Aderem) has extensive experience in macrophage biology and will allow the applicant to study the role of TOLLIP variation on phagocytosis and autophagy. Third, he will determine the breadth of the effects of TOLLIP on the immune system by studying how TOLLIP may affect dendritic cell maturation, T cell polarization, and cytokine responses. The applicant's long term goals are to seek a clinical appointment in a Division of Infectious Diseases at an academic center, develop sources of independent research funding, and devote >75% of his time to running a lab and collaborative clinical studies. Tuberculosis stimulates intense inflammatory response, but regulation of this response is key to control of infection. The regulation and nature of an optimal inflammatory response to Mycobacterium tuberculosis (MTb) remains poorly understood in humans. Insight into mechanisms of negative regulation of the TLR- mediated innate immune response to MTb could provide significant breakthroughs in the design of new vaccines and drugs. We hypothesized that TOLLIP and its common variants negatively regulate TLR signaling in human monocytes and are associated with susceptibility to tuberculosis. Using shRNA knockdown of TOLLIP in peripheral blood human monocytes, we found that TOLLIP suppresses TNF and IL-6 production after stimulation with TLR2 and TLR4 ligands. We also discovered a common polymorphism (rs5743899) that is associated with TOLLIP deficiency in a Caucasian population. Furthermore, in a case-population study in Vietnam with 760 cord-blood samples and 671 TB case patients, we found that the SNP rs5743899 was associated with susceptibility to tuberculosis (p=6.97x10-7). Together, these data demonstrate that TOLLIP has an anti-inflammatory effect on TLR signaling in humans and that TOLLIP deficiency is associated with an increased risk of TB. These data implicate an unexpected mechanism of negative regulation of TLR signaling in human TB pathogenesis. In this application, we will examine human variants of TOLLIP and their role in regulating macrophage, dendritic cell, and T cell responses to MTb infection. In Aim 1, I will determine the specific polymorphisms commonly found in the population that affect TOLLIP function and influence risk for developing tuberculosis. Furthermore, we will more fully characterize how TOLLIP influences phagocytosis, autophagy, and mycobacterial replication in macrophages and dendritic cells. I will also combine our human population studies with mechanistic studies in Tollip -/- mice. In Aim 2, I will use multiple techniques, including mouse studies, study of ex vivo infant cytokine response to BCG, and a unique case-control study of pediatric TB in South Africa to study the in vitro and in vivo effects of TOLLIP variation on dendritic cell biology and the effect of TOLLIP variation on the regulation of innate immune priming of the adaptive immune response. This work will be performed at the University of Washington School of Medicine in Seattle. The applicant and proposed mentor are in the Division of Allergy and Infectious Diseases, which consists of 73 fulltime faculty members whose total grant support exceeds $135 million annually. This Division is an ideal environment for training physician-scientists as more than 80% of past fellowship trainees have obtained faculty positions in academic medicine. The applicant proposed research takes advantage of his strong background in cellular immunology but requires additional training in molecular biology, innate immunology, and human genetics. He has detailed coursework, seminars, and meetings to address these needs and has developed metrics for assessing his progress. He has also assembled a scientific advisory panel to provide one-on-one expertise. Finally, the proposed mentor has a demonstrated track record with these techniques and has successfully mentored several other junior scientists. NARRATIVE: Understanding the immunologic and genetic factors that confer protection from tuberculosis is vital to the development of successful vaccines and therapeutics. TOLLIP is a recently discovered regulator of the Toll-like receptor pathway that is critical for an effective immune response to tuberculosis. I propose to focus on the mechanisms by which TOLLIP regulates innate immunity by determining the specific common variants that affect TOLLIP function. I will also examine the role TOLLIP plays in regulating cellular uptake and destruction of tuberculosis. Lastly, I will study how TOLLIP influences long term immune responses to BCG in infants. I will correlate this work with ongoing studies in Tollip-deficient mice This work will provide important information about the role of TOLLIP in influencing the immune response to tuberculosis, provide insight into the components of an effective immune response to infections, and may lead to improved vaccines and vaccine adjuvants to tuberculosis.